Genetics of the Bacterial Cell

Francois Jacob

If I find myself here today, sharingwith Andre Lwoff and Jacques Monodthis very great honor which is beingbestowed upon us, it is undoubtedlybecause, when I entered research in1950, I was fortunate enough to ar-rive at the right place at the righttime. At the right place, becausethere, in the attics of the Pasteur In-stitute, a new discipline was emergingin an atmosphere of enthusiasm, lucidcriticism, nonconformism, and friend-ship. At the right time, because thenbiology was bubbling with activity,changing its ways of thinking, discov-ering in microorganisms a new andsimple material, and drawing closer tophysics and chemistry. A rare mo-ment, in which ignorance could be-come a virtue.

Lysogeny and Bacterial Conjugation

The laboratory of Andre Lwoff wastraversed by a long corridor whereeveryone would meet for endless dis-cussions of experiments and hypoth-eses. At one end of the corridor,Jacques Monod's group was addingf,-galactosides to bacterial cultures toinitiate the biosynthesis of ,8-galacto-sidase; at the other end, Andre Lwoffand his collaborators were dousing cul-tures of lysogenic bacteria with ultra-violet -light, having just discovered that

W_A of initiating the biosynthesis ofbacteriophage. Each was therefore "in-ducing" in his own way, convincedthat the two phenomena had nothingin common, save a word.

Copyright © 1966 by the Nobel Foundation.The author is head of the department of

cellular genetics at the Pasteur Institute, Paris,France. This article is the lecture he delivered inStockholm, Sweden, 11 December 1965, when hereceived the Nobel Prize in Physiology or Medi-cine, which he shared with Andre Lwoff andJacques Monod. It is published here with thepermission of the Nobel Foundation and will alsobe included in the complete volumes of the Nobellectures in English, published by the ElsevierPublishing Company, Amsterdam and New York.

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Having come to prepare a doctoralthesis with Andre Lwoff, I was as-signed the study of lysogeny inPseudomonas pyocyanea. Thus Iconscientiously set out to irradiate thisorganism. However, it soon becameapparent that thie problem of lysogenywas primarily that of the relationshipbetween the bacterium and bacterio-phage, in other words, a matter ofgenetics.The genetics of bacteria and bacteri-

ophages was born just 10 years ear-lier, with a paper by Luria and Del-briick (1). It had continued to growwith the investigations of Lederbergand Tatum (2), Delbriick and Bailey(3), and Hershey (4). But this youngscience had already produced manysurprises for biologists. The most im-portant was the demonstration byAvery, McLeod, and McCarthy (5),and later by Hershey and Chase (6),that genetic specificity is carried byDNA. For the first time, it becamepossible to give some chemical and-physical meaning to the old biologicalconcepts of heredity, variation, andevolution. Such a molecular interpre-tation of genetic phenomena is exactlywhat was provided in the structure ofDNA proposed by Watson and Crick(7).Another surprise was the realization

that their rapid growth rate, their abilityto adapt to many different media, andthe variety of their mechanisms of ge-netic transfer make bacteria and vi-ruses objects of choice for studyingthe function and reproduction of thecell. The work of Beadle and Tatum(8), Lederberg (9), and Benzer (10)had shown that with a little imagina-tion one can exert on a populationof microorganisms such a selectivepressure as to isolate, almost at will,individuals in which a particular func-tion has been altered by mutation. In-deed, one of the most effective waysof determining the normal mechanisms

of the cell is to explore abnormali-ties in suitably selected monsters.The first attempts to anal-y-ze lysog-

eny genetically, intended to deter-mine the location of the prophage inthe bacterial cell, were carried out in1952 by E. and L. Lederberg (11) andby Wollman (12). Certain crosses be-tween lysogenic and nonlysogenic bac-teria suggested some linkage betweenthe lysogenic character-determined bythe A prophage of Escherichia coli-and other characters controlled by bac-terial genes. However, other crossesgave anomalous results. In fact, theanswer obtained from these experi-ments could hardly be decisive, sincethe mechanism of conjugation was notunderstood at the time.

It was with the intention of con-tinuing this study under somewhatdifferent conditions that I began towork with Elie Wollman; very soonour collaboration became a particular-ly close and friendly one. We wantedin the first place' to understand theanomalies observed in crosses betweenlysogenic and nonlysogenic bacteria, inparticular the fact that the lysogeniccharacter was not transmitted to re-combinants except when carried by thefemale. To study this problem, weused a mutant male bacterium recent-ly isolated by William Hayes andnamed Hfr, because, in crosses withfemales, it produced recombinantswith high frequency (13). Upon cross-ing such lysogenic Hfr males to non-lysogenic females, we were surprisedto find out that the zygotes formedby more than half the males happenedto lyse and produce phage (14). Thisphenomenon, termed zygotic induc-tion, showed that the equilibrium be-tween the prophage and the bacteriumis maintained by some regulatory sys-tem present in the cytoplasm of alysogenic bacterium but absent from anonlysogenic one. Moreover, it showedthat a genetic character transferred bythe male can be expressed in thezygote without being integrated intothe chromosome of the female bacte-rium. It thus became possible in bac-terial conjugation to distinguish ex-perimentally between transfer of ge-netic material and recombinationalevent.

In order to bring the analysis ofconjugation down from the level ofthe population to the level of the in-dividual bacterial pair, we had to un-derstand how the genetic material ofthe male is transferred to the female.

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In particular, one could try to inter-rupt conjugation after various times inorder to find out when the transfertakes place. Elie Wollman had thesomewhat startling idea of interruptingconjugation by placing a mating mix-ture in one of those blenders whichordinarily find service in the kitchen.It turned out that the shearing forcesgenerated in the blender separate malesfrom females; the male chromosomeis broken during transit, but the chro-mosomal fragment that has alreadypenetrated the female can express itspotentialities and undergo recombina-tion. In this way, it could be shownthat, after pairing, the male slowlyinjects its chromosome into the fe-male. This injection follows a strictschedule and, with any particularstrain, the injection always starts atthe same point (15). Marvelous or-ganism, in which conjugal bliss canlast for nearly three times the life-spanof the individual!With this system, it became rela-

tively easy to analyze the genetic con-stitution of E. coli, to show that bac-terial characters are arranged on asingle linkage group, termed the bac-terial chromosome, and to map them,not only by classical genetic methods,but also by physical and chemicalmeasurements. Moreover, two new in-sights emerged from this study. First,the bacterial chromosome turned outto be a closed, or circular, structure.Second, it was not as fixed a unit asone might have believed: other geneticelements, termed episomes (for exam-ple, a phage chromosome or a sexfactor), can be added to or sub-tracted from it (16). These propertieshappened to be of great value in sub-sequent studies of the bacterial celland of its functioning.

Expression of the Genetic

Material: The Messenger

In addition to its interest for theanalysis of strictly genetic phenomena,bacterial conjugation proved particu-larly well adapted for the analysis ofcellular functions, since it provided ameans of transferring to an entire pop-ulation a given gene at a given time.The phenotypic effects produced bythe sudden appearance of a new genein the recipient bacterium are thenmanifested without the accompanyingcomplications that occur in higher or-ganisms as a result of morphogenesisand cellular differentiation.10 JUNE 1966

At his end of the corridor, JacquesMonod had reached the conclusionthat further progress in the under-standing of enzymatic induction re-quired genetic analysis. Two types ofmutations which altered the inducedbiosynthesis of f8-galactosidase wereknown at that time. One type abol-ished the capacity to produce an ac-tive protein. The other changed the in-ducible character of enzyme synthesisso that it became constitutive, that is,able to proceed even in the absenceof a 8&-galactoside inducer. How arethese genes expressed? What is the re-lationship between the genetic de-terminants revealed by these muta-tions? What do the "inducible" or"constitutive" characters result from?Many of these questions could be ex-perimentally approached through bac-terial conjugation. By using male andfemale bacteria of suitable genotypes,one could transfer the desired allele ofa given gene into a bacterium, andthen study the conditions of enzymesynthesis in the zygote.

Such experiments were carried outin collaboration with Arthur Pardee,who had come to spend a year at thePasteur Institute (17). They led to twonew concepts. The first was relevantto the mechanism of induction itself.Transfer into a constitutive bacteriumof the genetic determinant for induci-bility of the enzyme by 8-galactosidesresulted in formation of transitory dip-loids, heterozygous for the characters"inducible/constitutive." Obviously, thephenotype of such zygotes should per-mit a choice among the different hypoth-eses then proposed to explain induc-tion. The experiments showed that the"inducible" allele can express itself in-dependently of the gene controllingthe synthesis of the enzyme; and thatit is dominant over the "constitutive"allele. This result revealed the existenceof a special gene which controls induc-tion by forming a cytoplasmic productthat inhibits synthesis of the enzymein the absence of inducer. As we shallsee later, this finding changed existingnotions about the mechanism of induc-tion and made possible a geneticanalysis of the systems which regulatethe rates of protein synthesis.The second observation concerned the

functioning of the genetic material. Bytransferring the gene that governs thestructure of a protein into a bacteriumwhich lacks it, one can determine theconditions under which this gene isexpressed in the zygote. Here again,different predictions could be made,

depending on the nature of the mech-anisms postulated for informationtransfer in the formation of proteins.From kinetic analysis of protein syn-thesis, one could expect informationconcerning the primary gene product,the time required for its synthesis, andits mode of action. The experimentsshowed that, once transferred into abacterium, and before genetic recom-bination has occurred, the gene con-trolling the structure of a protein canbegin to function without detectabledelay, producing protein at the maxi-mal rate.

This was quite a surprising observa-tion, for it was inconsistent with thenotions that were prevalent at the time.Gene expression was then usually be-lieved to consist in the accumulationof stable structures in the cytoplasm,probably the RNA of ribosomes,which were assumed to serve as tem-plates specifying protein structure (see18). Such a scheme, which can besummarized by the aphorism "onegene-one ribosome-one enzyme," washardly compatible with an immediateprotein synthesis at maximal rate.

Further study of this problem re-quired the withdrawal of a gene froma bacterium in order to examine theconsequences of this withdrawal onthe synthesis of the corresponding pro-tein: stable templates, if present, shouldpermit residual synthesis. But, althoughconjugation made it easy to inject aparticular gene, the extraction of aparticular gene from a whole bacterialpopulation appeared to be an impos-sible operation. What could be done,however, was to transfer a segmentof chromosome heavily labeled withp32 and then destroy the gene understudy by p32 decay. This delicate ex-periment was carried out by MonicaRiley in the laboratory of Arthur Par-dee; it showed unambiguously thatcapacity to produce the protein doesnot survive destruction of the gene(19).The answer was clear: gene expression

cannot proceed through formation ofstable templates. About the same time,the genetic and kinetic analyses of in-duction further strengthened this be-lief. Induction appeared to take placealmost instantaneously and to act onstructures which often specified sev-eral proteins, not merely a single one.This finding was likewise inconsistentwith the theories then current, sinceit did not fit with the observed homo-geneity of the ribosomes.By virtue of their stability, their

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homogeneity, and their base composi-tion, the two known species of RNAdid not fulfill the requirements forcytoplasmic templates. Since the notionthat synthesis of proteins could occurdirectly on DNA was incompatiblewith the cytoplasmic localization ofthe ribosomes and their role in thissynthesis, only one possible hypothesisremained: it was necessary to postu-late the existence of a third speciesof RNA, the messenger, a short-livedmolecule charged with transmission ofgenetic information to the cytoplasm(20). According to this hypothesis, theribosomes are nonspecific structures,which function as machines to trans-late the nucleic language, carried bythe messenger, into the peptidic lan-guage, with the aid of the transferRNA's. In other words, the synthesisof a protein must be a two-step proc-ess: the deoxyribonucleotide sequenceof DNA is first transcribed into mes-senger, the primary gene product; thismessenger binds to the ribosomes,bringing them a specific "program,"and the nucleotide sequence of the mes-senger is then translated into the aminoacid sequence. Despite the objectionsraised against it, this messenger hypoth-esis possessed two main virtues in oureyes: on the one hand, it alloweda coherent interpretation of a numberof known facts which had, until then,remained isolated or incompatible; onthe other hand, it led to some preciseexperimental predictions.

In fact, even before it had appearedin print, the messenger hypothesis re-ceived two experimental confirma-tions. Sydney Brenner and I had de-cided to spend the month of June1960 with M. Meselson, hunting forthe messenger in the laboratory ofMax Delbriick at the California Insti-tute of Technology. The best candidatefor the role of messenger seemed tous to be the RNA detected by Her-shey (21) and later by Volkin andAstrachan (22) in bacteria infected withT2 phage. Thanks to the extraordinaryintellectual and experimental agilityof Sydney Brenner, we were able toshow, within a few weeks, that theRNA formed by the phage associateswith ribosomes synthesized wholly be-fore infection, to produce on themphage proteins. The same ribosomescan thus make either phage or bac-terial proteins, depending on the mes-senger with which they associate. Ac-cordingly, it is the messenger whichbrings to the ribosomes a specific pro-gram for synthesis (23).

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At this time, another member of ourgroup at the Pasteur Institute, Fran-gois Gros, had gone to spend severalmonths at Harvard in the laboratoryof J. D. Watson. With their collabo-rators, they rapidly succeeded in dem-onstrating the existence of a messengerfraction in the RNA of growing bac-teria, and in establishing its principalproperties (24). The course of eventswhich led to the recognition and isola-tion of the messenger has been de-scribed by J. D. Watson (25).

Genetic Activity and Its

Regulation: The Operon

Experiments on genetic transfer byconjugation not only led to a revisionof the concepts on the mechanisms ofinformation transfer which occur inprotein synthesis; they also made itpossible to analyze the regulation ofthis synthesis.The most striking observation that

emerged from the study of phageproduction by lysogenic bacteria andof induction of p-galactosidase syn-thesis was the extraordinary degree ofanalogy between the two systems. De-spite the obvious differences betweenthe production of a virus and that ofan enzyme, the evidence showed thatin both cases protein synthesis is sub-ject to a double genetic determinism:on the one hand, by structural genes,which specify the configuration of thepeptide chains; on the other hand, byregulatory genes, which control the ex-pression of these structural genes. Inboth cases, the properties of mutantsshowed that the effect of a regulatorygene consists in inhibiting the expres-sion of the structural genes, by form-ing a cytoplasmic product which wascalled the repressor. In both cases, theinduction of synthesis (whether ofphage or of enzyme) seemed to resultfrom a similar process: an inhibitionof the inhibitor. Thus, to our surprise,these two phenomena, studied at op-posite ends of the corridor, appeaLredto share a common fundamental mech-anism. It should be emphasized thatthis analogy was invaluable to us. Inbiology, each material has its ownvirtues and is of particular valuefor a certain kind of experimental in-vestigation. The combination of twosystems significantly increased ourmeans of analysis.The existence of a specific inhibitor,

the repressor, had an immediate corol-lary: the protein-forming apparatus

must contain a site on which the re-pressor acts in order to block syn-thesis. The repressor itself could beregarded as a chemical signal emittedby the regulatory gene. The signalmust have a receptor. The receptorhad to be specific, hence geneticallydetermined, and hence accessible tomutation. In a system that permits in-duced biosynthesis of an enzyme, anymutation damaging one element of theemitter-receptor system which inhibitsthe synthesis should result in constitu-tive enzyme production. Consequentlyit seemed difficult to distinguish mu-tations affecting the emitter from thoseaffecting the receptor, until we rea-lized that the distinction should be rel-atively easy to make in a diploid. Thispoint can be illustrated by a simpleanalogy. Let us consider a house inwhich the opening of each of twodoors is controlled by a little radioreceiver. Let us suppose, furthermore,that somewhere in the vicinity thereexist two transmitters, each sendingout the same signal, which preventsthe opening of the doors. If one ofthese transmitters is damaged, theother continues to send out signals andthe doors remain closed: the damagedtransmitter can be considered as "re-cessive" with respect to the normalone. On the other hand, if one ofthe receivers is damaged, it no longerresponds to the inhibitory signal, andthe door which it controls (but onlythat one) opens. The damaged re-ceiver is thus "dominant" over thenormal one, but the lesion is mani-fested only by the door which it con-trols: the effect is cis and not trans,in genetic terminology (see 26).Thus it should be possible in prin-

ciple, by the use of diploid bacteria,to distinguish, among constitutive mu-tations, those due to the regulatorygene from those due to the receptor.In fact, phage mutants correspondingto one or the other of these types hadbeen known for a long time, althoughtheir nature became clear only in thelight of this scheme. The existence ofsuch mutations in phages encouragedus to search for analogous bacterialmutations affecting the enzymes of thelactose system. For that purpose, how-ever, diploid bacteria were required.Although conjugation allowed forma-tion of transitory diploids, their pro-duction was tricky and their analysiscomplicated. However, certain observa-tions which had recently been madeby Edward Adelberg led to the ideathat the sexual episome F, which gov-

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erns conjugation in E. coli, might un-der certain conditions incorporate andsubsequently replicate a small frag-ment of the bacterial chromosome(27). By using a series of strains inwhich the sexual episome was insertedat various points along the bacterialchromosome, we succeeded in isolatingepisomes which had incorporated aneighboring chromosomal fragment.Bacteria harboring such an episomebecome stable diploids for a small ge-netic segment, so that it is easy tomake all possible combinations of al-leles in this segment (28).Having -thus constructed the requi-

site genetic tool for our analysis, weset out to isolate under different con-ditions a whole series of mutants con-stitutive for the lactose system, in or-der to subject them to functional anal-ysis. These mutants proved to belongto two quite distinct groups, which pos-sessed the predicted properties for thetransmitter and the receiver, respec-tively.Many of these mutations were found

to be "recessive" with respect to thewild-type allele. They allowed a defi-nition of the transmitter, that is, ofthe regulatory gene. Some of these mu-tations possessed characteristic proper-ties which led to the indirect identi-fication of the repressor, the productof the regulatory gene (29). They arediscussed in greater detail in JacquesMonod's lecture.

In the second group, *the mutationsturned out to be "dominant" over thewild-type allele, and only those geneswhich were located on the same chro-mosome, that is, in cis position, wereexpressed constitutively. With thesemutations, it was possible to define thereceptor of the repressor, termed theoperator (30).The study of these mutants led,

furthermore, to the notion that in bac-teria the genetic material is organizedinto units of activity called operons,which are often more complex thanthe gene considered as the unit offunction. In fact, the lactose systemof E. coli contains three known pro-teins, and *the three genes governingtheir structure are adjacent to one an-other on a small segment of thechromosome with the operator at oneend (Fig. 1). Constitutive mutations,whether due to the alteration of theregulatory gene or of the operator, al-ways display the remarkable propertyof being pleiotropic; that is, they affectsimultaneously, and to the same extent,the production of the three proteins.10 JUNE 1966

The regulatory circuit therefore had toact on one integral structure contain-ing the information which specifiesthe amino acid sequences of thethree proteins. This structure could on-ly be either the DNA itself or a mes-senger common to the three genes.This idea was further supported by theproperties observed in mutations affect-ing the structural genes of the lactosesystem. Whereas some of these muta-tions obey Beadle and Tatum's "onegene-one enzyme" rule in the sense

messenger

that they abolish only one of the threebiochemical activities, others violatethis rule by affecting the expression ofseveral genes at a time (31, 32).The notion of the operon, a group-

ing of adjacent structural genes con-trolled by a common operator, ex-plained why the genes controlling theenzymes of the same biochemical path-way tend to remain clustered in bac-teria, as observed by Demerec andHartman (33). Similarly, it accountedfor the coordinate production of en-

fe -golactosideproteins

y Ac zFig. 1. The lactose region of Escherichia coli. The circle represents the E. coli chromo-some and shows the position of the lactose region (Lac) among other markers. Anenlargement of the lactose region is shown below. i, Regulatory gene; o, operator;p, promotor; z, structural gene for ,-galactosidase; y, structural gene for ,-galactosidepermease; Ac, structural gene for ,-galactoside transacetylase. The structural genesprobably synthesize a single messenger (of which the 5'-phosphate end is most likelyat the operator end) which associates with the ribosomes to form a polysome wherethe different peptide chains (of which the amino end probably corresponds to theoperator end) are synthesized. The regulatory gene produces a specific repressorwhich, acting at the level of the operator, blocks the production of the messengerand hence of the proteins. The ,-galactoside inducers act on the repressor to inactivateit, thus permitting the production of the messenger and consequenly of the proteinsdetermined by the operon.

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zymes already found in certain bio-chemical pathways (34). Although atfirst the operon concept was based ex-clusively on genetic criteria, it now in-cludes biochemical criteria as well.There are, in fact, a number of experi-mental arguments, both genetic (32, 35)and biochemical (36), in support of theinference that an operon produces asingle messenger, which binds to ribo-somes to form the series of peptidechains determined by the different struc-tural genes of the operon.We can therefore envision the ac-

tivity of the genome of E. coli asfollows. The expression of the geneticmaterial requires a continuous flow ofunstable messengers which dictate tothe ribosomal machinery the specific-ity of the proteins to be made. Thegenetic material consists of operonscontaining one or more genes, eachoperon giving rise to one messenger.The production of messenger by theoperon is, in one way or another, in-hibited by regulatory loops composedof three elements: regulatory gene, re--pressor, operator. Specific metabolitesintervene at the level of these loopsto play their role as signals: in in-ducible systems, to inactivate the re-pressor and hence allow production of

ChromosomeL_ _A&Y+ Z+ + iS

messenger and ultimately of proteins;in repressible systems, to activate therepressor, and hence inhibit productionof messenger and of proteins. Ac-cording to this scheme, only a frac-tion of the genes of the cell can beexpressed at any moment, while theothers remain repressed. The networkof specific, genetically determined cir-cuits selects at any given time thesegments of DNA that are to be tran-scribed into messenger and consequent-ly translated into proteins, as a func-tion of the chemical signals comingfrom the cytoplasm and from the en-vironment.From the beginning, the conception

of the genetic material as being formedof juxtaposed operons whose activityis regulated by a single operator siteentailed a precise experimental predic-tion: a chromosomal rearrangementwhich would separate some structuralgenes from their operator and linkthem to a different operon controlledby its own operator should place theactivity of these structural genes un-der a new regulatory control. But fora while, although some genes couldbe separated from their operators asa result of certain mutations, they be-came reattached to an unidentified re-

Pros Ph- T6R

Pur Gal

K T EO*- |-II -4-4. . -.I I - - - -- -II IIAAA

F Ay' z* o+ is Proe Ph+ T6S P,+o?

F Loc Pur

Aty+ z d3FOLac Pur

Fig. 2. Deletion fusing a fragment of the lactose operon and a fragment of apurine operon in E. coli. The upper part of the diagram represents the originalheterozygous diploid structure of a bacterium containing a sexual episome whichhas incorporated an important chromosomal fragment by recombination. In thecentral part of the diagram, the hatched region shows the zone eliminated by a dele-tion which occurred in the episome. The lower part of the diagram shows thestructure formed as a result of the deletion. The latter connected a termi-nal fragment of the z gene (determining 8-galactosidase) with an initial frag-ment of the Pur 8 gene (determining an enzyme of purine biosynthesis). A newoperon~is thus formed from the Pur a gene (determining one of the proteins ofpurine biosynthesis), a structure formed by part of the Pur pa gene and part of thez gene (probably producing a hybrid peptide chain consisting at the amino end ofa Pur p sequence and at the carboxyl end of a sequence from z), the y gene (deter-mining 8-galactoside permease), and the Ac gene (determining 8-galactoside trans-acetylase). The expression of the operon is repressed by purines, presumably at thelevel of a purine operator, which is itself sensitive to a repressor specifically activatedby purines (39).

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gion of the chromosome and conse-quently subject to an unknown sys-tem of regulation which remained be-yond our experimental reach (37,38).

Only recently has it become possi-ble to obtain a fusion of the lactoseoperon of E. coli with another knownoperon, by using bacteria which werediploid for the chosen region (39). Atpresent, we know only a limited num-ber of genes on the bacterial chro-mosome and a still more limited num-ber of genes whose activity can bemodified by the action of external me-tabolites. Any deletion which fuses twoof these regions is likely to be relative-ly large and therefore to include agene whose product is required forgrowth or division; it would thus belethal in a haploid bacterium. In dip-loid bacteria, on the other hand, ithas been possible to isolate a seriesof deletions covering about 50 to 80genes; at one end, these -deletionsterminate in the gene controlling thestructure of pl-galactosidase, and at theother, in different regions of the chro-mosome. Some terminate in one ofthe two cistrons belonging to a purineoperon, while leaving the other cis-tron intact (Fig. 2).

In these mutants, synthesis of thetwo proteins of the lactose region de-termined by the -two genes left intactby the deletion is no longer inducibleby f3-galactosides. Such a result couldbe predicted since the deletion has de-stroyed both of the elements (regulatorygene and operator) responsible for thespecific regulation of the lactose sys-tem. But this synthesis has become re-pressible by the addition of purines.It is thus clear that, in the deletion,the fragment of the lactose operon andthe fragment of the purine oneronlhave been fused to form a new operonwhich, from all appearances, producesa single messenger containing the ge-netic information for the synthesis ofthe proteins involved both in the bio-synthesis of purines and in the utiliza-tion of lactose. But the system whichdetermines the regulation of this mes-senger must be the operator of thepurine region, sensitive to a repressoractivated by purines.

In the same way, deletions fusingthe lactose operon with the operon con-trolling tryptophan biosynthesis haverecently been isolated (40). The expres-sion of the genes of the lactose operonwhich are still intact has consequent-ly become repressible by tryptophan.

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KI

a III

b IIIII

c IId I a~

e I1

f I

Ka I

b I

I I

Fig. 3. Schematic diagram showing achange of polarity, and hence of strand,in an inversion.

The type of regulation imposed onthe expression of the genes belongingto a given operon thus depends ex-clusively on the operator, that is, insome way, on the nucleotide sequencelocated at the proximal extremity ofthe operon. In this manner, both thenature of the metabolites on whichregulation depends and the inducibleor repressible character of the regu-lation are determined by the respectivepositions of the genes along the chro-

Ac y z DOI 1 --- DNA

A JVVVVVAAf-A m- RNAVZOMMW aVZW@a 7tEM VW protein

Ac y z p

Ac y z p o

c wv-v

Fig. 4. The three possible models for thefunctioning of 'an operator. A, The opera-tor (o) is transcribed and translated intoprotein. B, The operator is transcribedbut not translated. C, The operator isneither transcribed nor translated. Ob-viously, depending on whether or not theoperator is translated or is transcribed,the repressor could act at the level of theprotein, of the messenger, or of the DNAitself.

10 JUNE 1966

mosome and, more particularly, bytheir association with a particular opera-tor segment. Obviously, it is the asso-ciations most favorable to the organismthat are selected.The presence of units of activity

and regulation constituted by polycis-tronic operons implies the existence ofa double system of punctuation in thenucleic text. One type of punctuationmust permit the "slicing" of the longDNA duplex into "sections of tran-scription" corresponding to operons:this must serve as the point of recog-nition for the RNA polymerase, toshow it not only where to start andfinish the transcription of an operon,but also which strand of DNA is to betranscribed. Under certain conditions,one can obtain transpositions of the lac-tose operon into a region of the chro-mosome different from the normal re-gion, and these insertions can be ori-ented in either one direction or theother (40). It should be noted that, incase of an inversion, the 3', 5' polarityof the DNA strands requires that thesequence to be transcribed into mes-senger must change with respect notonly to direction, but also to strand(Fig. 3). For the insertions obtained,the lactose operon seems to be equallywell expressed whether or not therehas been an inversion. Consequently,it must be assumed that (i) all thegenetic information of E. coli is notnecessarily contained in the samestrand of DNA; (ii) a genetic signalought to indicate the start of theoperon, as well as the direction oftranscription; and- (iii) another signalought to mark the end of the operon.If two operons of opposite orientationsoccur in juxtaposition, in the absenceof a signal "end of transcription," thetranscription of one operon couldeventually proceed along the other, inthe DNA strand which is not normallysupposed to be transcribed.The second punctuation mark of

the nucleic text must, at the time oftranslation, allow the "slicing" of themessenger into the various peptidechains corresponding to the respectivegenes of the operon. This punctua-tion serves as a signal to the transla-tion system (ribosomes, sRNA, andso on) to delimit the amino terminaland the carboxy terminal ends of eachpeptide chain.

In the lactose system of E. coli, theanalysis of a series of deletions showsthat the operator is situated outsidethe first known structural gene of the

operon (41, 38), from which it appearsto be separated by a region called thepromotor, which is indispensable to theexpression of the entire operon (37).The promotor probably corresponds toone of the punctuation marks, eitherfor transcription or for translation.There are reasons to believe that theoperator itself is not translated into apeptide chain, but we still do not knowwhether it is transcribed into messen-ger and whether the repressor acts atthe level of the messenger or of theDNA itself (Fig. 4). It is not possibleto discuss in detail here the experi-mental arguments or the hypotheses(20, 32, 42) concerning the site of ac-tion of the repressor. However, theensemble of results recently obtainedin various laboratories (43) indicatesthat synthesis of the messenger fromits 5'-phosphate terminus, like that ofthe first peptide chain from its aminoterminus, both begin at the operatorend of the operon. The simplest hypoth-esis compatible with the results ofgenetic analysis, particularly with thestudy of deletions covering differentsegments of the operator region, isthat the promotor represents the punc-tuation of transcription, providing thesignal for RNA polymerase to startthe synthesis of the messenger for thisoperon on one of the two DNAstrands. If this is correct, the operatoris not transcribed into messengerand repression can be exerted only atthe level of DNA. This is the inter-pretation that now seems the mostplausible to the geneticist; but it isclear that, as usual, the last word willbelong to the chemist.

Organization of Genetic Material

in the Bacterial Cell: The Replicon

Genetic analysis had revealed thelogic of the circuits involved in theregulation of protein synthesis byshowing that these circuits are com-posed of elements which can be con-nected in different ways to respondto the needs of the cell. It seemedreasonable to assume that analogouscircuits, constructed on similar princi-ples and using similar elements, mightparticipate in other aspects of cellularregulation, and in particular to directthe replication of DNA in coordina-tion with cellular division.The systems involved in the replica-

tion of DNA seem to function moresubtly in the cell than when isolated in

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the test tube. There are a number ofexperimental arguments in favor of asemiconservative mechanism of repli-cation, as predicted by Watson andCrick from their model. Thanks tothe work of Kornberg (44) and hiscollaborators an enzyme is knownwhich can polymerize deoxyribonu-cleotides in the order dictated by thesequence of a piece of DNA servingas template. However, if a fragmentof bacterial DNA is transferred intoa recipient bacterium by transforma-tion or incomplete conjugation, thisfragment is incapable of replicating byitself. It can replicate only when inte-grated by recombination with one ofthe genetic structures in the host bac-terium.

In bacteria, the DNA is organizedinto much simpler units than those ob-served in the cells of higher organ-isms. The essential information forthe growth and division of the bac-terium is carried by a single element,the so-called "bacterial chromosome."In addition. other nonessential ele-ments, the "episomes.' may be intro-duced into the bacterial cell (16).

Many different kinds of work haverevealed that the best known of thesegenetic elements, the chromosome, be-haves genetically, structurally, andbiochemically as a single, integratedelement. It seems to consist of onedouble-stranded chain of DNA, veryprobably closed or circular (45). Repli-cation appears to start at a fixed pointon the molecule, and to continue regu-larly until the point of departure isreached again (45, 46). Under normalgrowth conditions, a new round ofreplication cannot begin until com-pletion of the previous one (47).

Although other bacterial genetic ele-ments are less well understood, theirproperties seem to be analogous. Thegenetic equipment of a bacterium canthus be considered to consist of dis-tinct struLctures, each containing a"molecule" of DNA which is circularaind of variable length.

Together with Sydney Brenner, wehave tried to explain the regulation ofDNA synthesis by means of circuitsresembling those involved in the con-trol of protein synthesis (48). We havebeen led to postulate that each genetic

element constitutes a unit of replica-tion or replicon, which determines acircuit controlling its own replica-tion in coordination with cell division.This hypothesis carries with it threedistinct predictions.

1) If each element contains somegenetic determinants controlling itsown replication, it should be possibleto isolate mutants in which the regu-latory circuit is impaired. In fact, foreach of the three elements examnined-bacterial chromosome, sexual epi-some, and phage-mutations can beobtained which abolish replication ofthe mutated element but not of others(49). The nature and properties ofthese mutations suLggest that they mod-ify a diffusible product which normal-ly acts on a punctuation mark of thereplicon, that is, on a particular nui-cleotide sequence, to permit the startof replication. Once the reaction is in-itiated, the system replicates the en-tire sequence attached to this punctua-tion.

Here again, a genetically deterniinedregulatory loop appears to operate. But,whereas in the synthesis of proteins

Fig. 5 (left). Sections of Bacillius subtilis. The nticlear body (N) is botind to the membrane by means of a mesosome (Ai!)(50). Fig. 6 (right). Sections of B. suibtilis placed for 30 minutes in O.5M sticrose. The mesosomes have been ptushed ouit ofthe cytoplasm. Duiring their retraction, they pull with them the nticleair bodies (N), which then atppear bouind directly to themembrane (50).

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the regulation seems to be negativeor repressive, in the synthesis of DNAthe available evidence indicates thatthe regulation involves a positive ele-ment, that is, one which acts on theDNA to trigger replication.

2) The circumstances of bacterialconjugation can be best interpreted byassuming that the sexual episome isattached to the bacterial membranenear the zone through which themale chromosome passes during mat-ing. Furthermore, to explain, duringbacterial growth, the segregation ofDNA after replication and the distri-bution of the two DNA copies in thetwo bacteria formed by cellular divi-sion, the simplest hypothesis consistsin assuming that all cellular repliconsare attached to the bacterial mem-'brane. It is the synthesis of the mem-brane between the points of attach-ment of the two DNA copies thatwould insure their normal segregation.The latter prediction appears to be

confirmed by Antoinette Ryter's elec-tron microscopic study of the "nu-clear bodies" in Bacillus subtilis (50).Each of these nuclear bodies seemsto be attached to a "mesosome," astructure formed by an invagination ofthe membrane (Figs. 5 and 6). Fur-thermore, by staining the membranewith a tellurium salt, it can be shownthat membrane synthesis does not takeplace uniformly over the bacterial sur-face, but rather in particular zonesclose to the attachment sites of thenuclear bodies. It is thus the growthof the membrane which seems to bringabout the segregation of the DNAelements formed by replication. Final-ly, Francois Cuzin has succeeded indemonstrating that two independentreplicons, such as the chromosomeand the sex factor, do not segregateindependently but remain associatedduring bacterial multiplication (51);presumably, each of these structuresis linked to the same element, possi-bly the same fragment of membrane.This fragment which might remainintact during growth and bacterial di-vision would thus constitute the actualunit of segregation, analogous to achromosome in a higher organism.

3) To explain the coordination be-tween the replication of DNA andthe growth and subsequent divisionof the bacterial cell, it must be as-sumed that DNA replication and itsregulation occur in the membrane. Thisseems to be indicated by certain phe-nomena observed during bacterial con-

10 JUNE 1966

Fig. 7. Schematic diagram of DNA replication in bacteria. The bacterium representedcontains two independent units: the "chromosome" and the sexual episome F. Thesetwo replicons are shown attached to the membrane at two distinct sites. At a givenpoint in the division cycle, the membrane transmits to each replicon a signal per-mitting replication to start. Replication progresses linearly, each replicon turningslowly across the membrane in which the enzymatic complex responsible for replica-tion is thought to occur. Two copies of each replicon are thus formed and are assumedto be attached to the membrane side by side. Membrane synthesis is considered tooccur between these regions to which the two copies of each replicon are attached,thus drawing them to either side, with the septum forming in the median region.No new round of replication is allowed until the membrane, having returned to itsoriginal state following cell division, transmits a new signal. The process is simplifiedin the sense that: (i) since the replication of DNA occurs one cycle ahead of divi-sion, bacteria generally have from two to four nuclear bodies (and not from oneto two); and (ii) each step is considered to be completed before the start of thenext one (48).

jugation: presumably a surface reactionwhich occurs while the male and fe-male come in contact triggers, in someway, a round of replication in themale. One of the structures thus syn-thesized remains in the male, whilethe other is progressively driven, dur-ing its formation, into the female (52).Furthermore, the idea that DNA syn-thesis occurs in the membrane has re-cently received some biochemical sup-port (53).Thus one comes to envision the ge-

netic equipment of bacteria as formedof circular DNA "molecules" consti-tuting independent units of replica-tion. These units would be associatedwith one membrane element, whichwould coordinate their replicationwith cell growth by means of regula-tory circuits (Fig. 7). The basic ge-netic information would be containedin the longest of these units, but sup-plementary information could be addedby the fixation of other replicons tothe membrane. It seems that one ofthe important steps in the passagefrom the cellular organization of pro-caryotes to that of eucaryotes in-volves invaginations of membranousstructures followed by differentiationinto specialized organelles (mitochon-dria, genetic apparatus) whose func-tions were originally carried out bythe bacterial membrane.

It is remarkable that the study of

the bacterial cell leads us to attributeto the membrane such an importantrole in the coordination of growth andcell division, since a similar conclu-sion has been reached on the basisof the study of cells from higher or-ganisms. In such cells, the processof morphogenesis and contact phe-nomena among cells indicate that thecell surface must also control cellularmultiplication by means of signalstransmitted, in one way or another,to the nucleus. Despite the evidentcomplexity of such cells in compari-son with bacteria, it must be assumedthat evolution has conserved a systemof molecular communication betweencell surface and DNA.

Conclusions

The two chemical activities of DNA,transcription, the copying of a singlestrand into a ribonucleotide sequence,and replication, the copying of bothstrands into deoxyribonucleotide se-quences, are controlled by a net-work of specific molecular interactionsdetermined by the genes. The mes-sage inscribed in the genetic materialthus contains not only the plans forthe architecture of the cell, but alsoa program to coordinate the syntheticprocesses, as well as the means of in-suring its execution.

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Perhaps one of the most importantcontributions of microbial genetics isthe definitive answer given to the oldproblem of the interaction betweengenes and cytoplasm, between heredityand environment. Although the demon-stration that acquired characters can-not be inherited had already beenmade by classical genetics, the expla-nation for this fact is now providedby the nature of the nucleic messageand of the genetic code. It is clear, onthe other hand, that the expression ofthe genetic material is subject to ex-ternal influences. Ten years ago, it stillseemed possible that, in certain proc-esses such as the induced biosynthesisof enzymes or of antibodies, the pres-ence of specific compounds could modi-fy the synthesis of proteins, moldtheir configurations, and hence altertheir properties. The environmentseemed to exercise an instructive ac-tion on the genes, to use Lederberg'sexpression (54), and thus to modulate-the sense of the genetic text. What thestudy of regulatory circuits has shownis that the compounds in questionserve only as simple stimuli: they actas signals to initiate a synthesis whosemechanism and final product remainentirely determined by the nucleotidesequence of the DNA. If the nucleicmessage may be compared with thetext of a book, the regulatory networkdetermines which pages are to be readat any given time. In the expressionof the nucleic message, as well as inits reproduction, adaptation resultsfrom an elective rather than an instruc-tive effect of the environment.Of course genetic analysis can do

no more than indicate the existence ofregulatory circuits. A chemical analy-sis, which should disclose the specificmolecular interactions, remains to bemade. No repressor has yet been iso-lated, and the nature of the complexesthat it can form with an operator or ametabolite remains obscure. We do notknow how molecules find each other,recognize each other, and combine toconstitute the regulatory network or toform such cellular superstructures asa membrane, a mitochondrion, or achromosome. We do not know howmolecules transmit the signals which

modify the activity of their neighbors.What is clear, however, is that the prob-lems to be solved by cellular biologyand genetics in the years to come tendincreasingly to merge with those inwhich biochemistry and physicalchemistry are involved.

Acknowledgment

The work summarized here could not havebeen pursued without the help of so many col-laborators that it is impossible for me to citethem without risking the injustice of unintentionalomissions.

This work has been supported by successivegrants from the Centre National de la RechercheScientifique, the Delegation Generale A la Re-cherche Scientifique et Technique, the Commis-sariat at l'Energie Atomique, the Fondation pourla Recherche m6dicale frangaise, the Jane CoffinChilds Memorial Fund, and the National ScienceFoundation.

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Genetics of the Bacterial CellFrancois Jacob

DOI: 10.1126/science.152.3728.1470 (3728), 1470-1478.152Science 

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